Antigen presentation is an obligatory step in the development of specific antibody and cellular immune responses that protect individuals from infection. Understanding this process should lead to novel approaches to control immune responses including the development new and more effective vaccines. The theme of this project remains the analysis of how antigens (Ags) are processed and presented to T lymphocytes. The central hypothesis of this project is that class I Ag processing involves the cleavage of Ags into peptides through the action of intracellular proteanases. There are 3 specific Aims: 1. What is the role of ubiquitin and the 265 proteasome in class I Ag presentation? The goals of this Aim are: (i) To evaluate whether and to what extent Ub and the 26S proteasome play a role in the processing of endogenous Ags for class I presentation; and, (ii) To test the hypothesis that the rate of degradation of Ags in the cytosol is a critical parameter that influences the efficiency of class I presentation. The experimental approach is to analyze whether blocking or enhancing degradation by the Ub-proteasome pathway of degradation affects class I-restricted Ag presentation. These studies will define the role of a major catabolic pathway in Ag processing. In addition, these experiments will define how degradation may control the immunogenicity of intracellular Ags and could provide a rationale strategy for designing more active vaccines. 2. Does a form of the proteasome generate appropriate peptides for class I presentation? The goal of this Aim is to examine whether the proteasome generates antigenic peptides and whether its activity/specificity is immunologically-regulated. The experimental approach will be to analyze in cell free systems the activity and specificity of the various forms of the proteasome from normal, mutant or interferon-stimulated cells. These experiments will define whether the 205 or 265 proteasome produce one or both appropriate cleavages for immunogenic peptides and what role the MHC- encoded subunits may play in this process. 3. How does a subset of APCs process exogenous Ags for MHC class I presentation? The goal of this Aim is to define the apparently novel pathway through which some APCs processes exogenous Ag. The experimental approach will use mutant (targeted gene disruption) and cloned-lines of these APCs in studies that will directly follow the fate of Ag and in experiments using modified-Ags and selective inhibitors to probe the key steps in this pathway.